Summary: Mast cell (MC) activation through the aggregation of cell surface IgE receptors (Fc?RI) leads to life- threatening conditions such as anaphylaxis and asthma. Recent exciting development in MC biology has been the discovery that they express a novel G protein coupled receptor (GPCR) known as MRGPRX2 (mouse counterpart MrgprB2), which contributes to a growing list of conditions such as pseudoallergy, neurogenic inflammation, non-histaminergic itch, allergic contact dermatitis and atopic dermatitis. The main objective of this proposal is to modulate Fc?RI and MrgprB2-mediated responses by targeting novel signaling pathways in MCs. Following their activation, most GPCRs undergo desensitization via their phosphorylation by GPCR kinases (GRKs) and the recruitment of the adapter proteins, ?-arrestins (?-arrs). We made four novel observations, which provide the basis of this proposal. First, we found that unlike most GPCRs, MRGPRX2 is resistant to GRK2-mediated desensitization but it contributes to IgE-mediated MC degranulation and cytokine production. Second, ?-arr2 inhibits both IgE and MrgprB2-mediated MC degranulation by modifying unknown components downstream of Ca2+ mobilization. Third, ?-arr2 contributes to MC chemotaxis in vitro and allergic contact dermatitis in vivo. Fourth, the effect of ?-arr2 on MC chemotaxis is associated with Ser3 dephosphorylation of the actin depolymerization factor cofilin. While activation of protein kinase C? (PKC?) promotes MC degranulation, PKC? phosphorylates cofilin at Ser23 and Ser24 to increase actin polymerization, resulting in cessation of degranulation. Based on these findings, we hypothesize that GRK2 promotes IgE- mediated responses in MCs via the regulation of Syk/Akt/NF-?B signaling but ?-arr2 modulates both Fc?RI and MrgprB2 responses by functioning as a scaffolding protein for phosphorylation and dephosphorylation of cofilin. In aim 1, we will determine the role of GRK2 on Fc?RI-mediated MC degranulation and cytokine generation in vitro and allergic response in vivo. In aim 2, we will determine the role of ?-arr2 on Fc?RI and MrgprB2- resposnes in vitro and inflammatory responses in vivo. Completion of this study may provide better rationale for the development of novel therapeutics for the MC-mediated disorders.